Methods to detect cancers more accurately or at earlier stages could lead to better outcomes for many cancer patients, including patents with pancreatic cancer. The success of this goal is facilitated by technologies that allow the rapid profiling and characterization of candidate biomarkers. Many technologies for that purpose are in development, each with unique optimal applications and advantages and disadvantages. Affinity-based methods, using antibodies or other affinity reagents, are preferred in applications where reproducible, specific and relatively high-throughput protein detection is required. The value of affinity-based methods has been enhanced through the use of microarrays, which allow multiplexed and high-throughput protein analysis in low sample volumes.
Pancreatic cancer is typically diagnosed at a late stage. The late stage detection combined with few treatment options lead to five year survival rates of less than 5%. Yeo, C. J., Cameron, J. L., Lillemoe, K. D., Sitzmann, J. V., Hruban, R. H., Goodman, S. N., Dooley, W. C., Coleman, J., and Pitt, H. A. Pancreaticoduodenectomy for cancer of the head of the pancreas. 201 patients. Ann Surg, 221: 721-731; discussion 731-723, 1995.
Because established disease can be difficult to diagnose due to clinical similarities with certain benign diseases such as chronic pancreatitis [2], some patients may receive sub-optimal treatment. Current diagnostic modalities include non-invasive imaging, endoscopic ultrasound, and cytology based on fine-needle aspiration [3]. These methods are useful for identifying pancreatic abnormalities and rendering an accurate diagnosis in many cases, but they come with high cost, significant expertise required for interpretation, and inherent uncertainty.
Blood-based diagnostic tests for pancreatic would be especially valuable because of the potential for routine and inexpensive screening. Several serum markers previously have been investigated for pancreatic cancer diagnostics. The CA 19-9 antigen—a carbohydrate blood group antigen—is elevated in 50-75% of pancreatic cancer cases and is typically used to confirm diagnosis or to monitor a patient's progress after surgery. Riker, A., Libutti, S. K., and Bartlett, D. L. Advances in the early detection, diagnosis, and staging of pancreatic cancer. Surgical Oncology, 6: 157-169, 1998. CA 19-9 is not used for early screening since it is not present in patients with certain blood types and is often elevated in benign disease.
Other carbohydrate antigens are associated with pancreatic cancer, such as SPAN-1 (Frena, A. SPan-1 and exocrine pancreatic carcinoma. The clinical role of a new tumor marker. Int J Biol Markers, 16: 189-197, 2001); DUPAN-2 (Kawa, S., Oguchi, H., Kobayashi, T., Tokoo, M., Furuta, S., Kanai, M., and Homma, T. Elevated serum levels of Dupan-2 in pancreatic cancer patients negative for Lewis blood group phenotype. Br J Cancer, 64: 899-902, 1991); CEA; CA-50; 90K (Gentiloni, N., Caradonna, P., Costamagna, B., E'Ostilio, N., Perri, V., Mutignani, M., Febbraro, S., Tinari, N., Iacobelli, S., and Natoli, C. Pancreatic juice 90K and serum CA 19-9 combined determination can discriminate between pancreatic cancer and chronic pancreatitis. Amer. J. Gastroenterology, 90: 1069-1072, 1995); CA 195 (Hyoty, M., Hyoty, H., Aaran, R. K., Airo, I., and Nordback, I. Tumour antigens CA 195 and CA 19-9 in pancreatic juice and serum for the diagnosis of pancreatic carcinoma. Eur J Surg, 158: 173-179, 1992); TUM2-PK (Oremek, G. M., Eigenbrodt, E., Radle, J., Zeuzem, S., and Seiffert, U. B. Value of the serum levels of the tumor marker TUM2-PK in pancreatic cancer. Anticancer Res, 17: 3031-3033, 1997); and CA 242 (Pasanen, P. A., Eskelinen, M., Partanen, K., Pikkarainen, P., Penttila, I., and Alhava, E. Multivariate analysis of six serum tumor markers (CEA, CA 50, CA 242, TPA, TPS, TATI) and conventional laboratory tests in the diagnosis of hepatopancreatobiliary malignancy. Anticancer Res, 15: 2731-2737, 1995).
Certain changes that occur in the sera of pancreatic cancer patients reflect the high level of inflammation associated with the disease. Pro-inflammatory cytokines, such as IL-6 and IL-8 (Wigmore, S. J., Fearon, K. C., Sangster, K., Maingay, J. P., Garden, O. J., and Ross, J. A. Cytokine regulation of constitutive production of interleukin-8 and -6 by human pancreatic cancer cell lines and serum cytokine concentrations in patients with pancreatic cancer. Int J Oncol, 21: 881-886, 2002), and the acute phase reactant C-reactive protein (CRP) are usually elevated in the sera of pancreatic cancer patients. Fearon, K. C., Barber, M. D., Falconer, J. S., McMillan, D. C., Ross, J. A., and Preston, T. Pancreatic cancer as a model: inflammatory mediators, acute-phase response, and cancer cachexia. World J Surg, 23: 584-588, 1999. Numerous other proteins have been evaluated as serum biomarkers for pancreatic cancer. The performance of tests based on single markers so far has not been good enough to be recommended for clinical application.
Prior studies were performed measuring one protein at a time, using sample and reagent volumes that in most cases prohibited large-scale studies of multiple candidate markers. The measurement of many putative cancer-associated serum proteins together, as enabled by antibody microarrays, has valuable uses. Multiple candidate markers are efficiently screened, allowing a broad characterization of the types of alterations present in cancer sera, and multiple measurements can be used in combination to potentially improve the diagnostic accuracy. Multiple, independent markers may be grouped together to improve diagnostic performance if the markers contribute complementary, non-overlapping discrimination information. The challenge for pancreatic cancer diagnostics is to find the particular protein alterations or combinations of protein alterations that usually occur early in cancer development and that do not occur in benign conditions.
The application of antibody and protein microarray methods to cancer research has been demonstrated in studies on proteins in sera, cell culture, and resected tissue samples. Miller, J. C., Zhou, H., Kwekel, J., Cavallo, R., Burke, J., Butler, E. B., Teh, B. S., and Haab, B. B. Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics, 3: 56-63, 2003; Huang, R.-P., Huang, R., Fan, Y., and Lin, Y. Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system. Anal. Biochem., 294: 55-62, 2001; Huang, R., Lin, Y., Shi, Q., Flowers, L., Ramachandran, S., Horowitz, I. R., Parthasarathy, S., and Huang, R. P. Enhanced protein profiling arrays with ELISA-based amplification for high-throughput molecular changes of tumor patients' plasma. Clin Cancer Res, 10: 598-609, 2004; Zhou, H., Bouwman, K., Schotanus, M., Verweij, C., Marrero, J. A., Dillon, D., Costa, J., Lizardi, P. M., and Haab, B. B. Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements. Genome Biology; 5: R28, 2004; Hamelinck, D., Zhou, H., Li, L., Verweij, C., Dillon, D., Feng, Z., Costa, J., and Haab, B. B. Optimized normalization for antibody microarrays and application to serum-protein profiling. Mol Cell Proteomics, 2005; Sreekumar, A., Nyati, M. K., Varambally, S., Barrette, T. R., Ghosh, D., Lawrence, T. S., and Chinnaiyan, A. M. Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins. Cancer Research, 61: 7585-7593, 2001; Lin, Y., Huang, R., Cao, X., Wang, S. M., Shi, Q., and Huang, R. P. Detection of multiple cytokines by protein arrays from cell lysate and tissue lysate. Clin Chem Lab Med, 41: 139-145, 2003; Knezevic, V., Leethanakul, C., Bichsel, V. E., Worth, J. M., Prabhu, V. V., Gutkind, J. S., Liotta, L. A., Munson, P. J., Petricoin, E. F. I., and Krizman, D. B. Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics, 1: 1271-1278, 2001; Tannapfel, A., Anhalt, K., Hausermann, P., Sommerer, F., Benicke, M., Uhlmann, D., Witzigmann, H., Nauss, J., and Wittekind, C. Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays. J Pathol, 201: 238-249, 2003; Hudelist, G., Pacher-Zavisin, M., Singer, C. F., Holper, T., Kubista, E., Schreiber, M., Manavi, M., Bilban, M., and Czerwenka, K. Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue. Breast Cancer Res Treat, 86: 281-291, 2004.
Alterations to Post-Translationally-Modified Proteins
Many proteins are modified through glycosylation, or the attachment of carbohydrate chains at specific locations. The structures of these chains are precisely regulated and often play a major role in protein function. Glycosylation is an important determinant of protein function, and changes in glycosylation are thought to play roles in certain disease processes, including cancer. Thus, the ability to efficiently profile and measure variations in glycosylation on multiple proteins and in multiple samples is valuable to identify disease-associated glycans alterations and new diagnostic markers. Specifically, the ability to efficiently profile the variation in glycosylation could lead to the identification of disease-associated glycan alterations and new diagnostic biomarkers.
The current methods for analyzing glycans are either cumbersome or very low throughput and not reproducible enough for diagnostics research. Traditionally, glycan structure is studied by enzymatic or chemical cleavage of carbohydrate groups, followed by gel or chromatography analysis and perhaps mass spectrometry analysis. While these methods are useful for determining glycan structures, they are not suitable for studies requiring reproducible measurements over many different samples or proteins, or for determining variation between populations of samples.
Affinity chromatography methods have been used to measure abundances of glycans. Useful affinity reagents for carbohydrate research are lectins—plant and animal proteins with natural carbohydrate binding functionality. Lectins have been used in a variety of formats such as affinity chromatography to isolate glycoproteins and modified ELISAs. Lectins and antibodies against carbohydrate epitopes have been used to identify cancer-associated glycosylation, although those methods do not identify which proteins are carrying the epitopes. Affinity chromatography methods could be coupled to immunoprecipitation methods to measure glycans on specific proteins.
As discussed below, the inventor has developed a high throughput affinity-based method that is practical for multiplexed studies. The inventor has applied the method of the present invention to the study of changes in glycan levels on serum proteins in pancreatic cancer patients. Further, the inventor has identified various biomarkers that, alone or in combination, are useful in methods of diagnosing pancreatic cancer including methods of differentiating pancreatic cancer from other pancreatic diseases.